The purpose of the study was to explore the immunological components that are responsible for the proliferative alterations in the different forms of retinal detachment (RD).n = 30), PVR (n = 16) and proliferative diabetic retinopathy (PDR) with tractional RD (n = 8). Vitreous fluids were obtained from 19 eyes with epiretinal membrane (ERM), which were used as control samples. A multiplex chemiluminescent immunoassay was performed to evaluate the concentrations of 48 cytokines, chemokines and growth factors.Vitreous fluids were collected during 23G pars plana vitrectomy from 54 eyes of 54 patients with different RD types, such as rhegmatogenous RD (RRD) without proliferative vitreoretinopathy (PVR) (The expression levels of eotaxin, IFN-gamma, IL-6, IL-8, IL-16, MCP-1, MIF and MIP-1 beta were significantly higher in all RD groups than in the ERM group. The levels of CTACK, IP-10, SCGF-beta, and SDF-1 alpha were significantly higher in patients with diabetic tractional RD and PVR than in other patients. The upregulation of VEGF and IL-18 was detected in PDR.Our results indicate that complex and significant immunological mechanisms are associated with the pathogenesis of different forms of RD: selected cytokines, chemokines and growth factors are upregulated in the vitreous of eyes with RD. The detected proteins are present in different concentrations both in RRD and PVR. In the presence of PVR and PDR, the majority of cytokines are upregulated; thus, they may serve as biomarkers to estimate the progression or severity level of proliferation and later to develop personalized therapeutic strategies to slow down or prevent pathological changes. Retinal detachment (RD) can cause vision loss if untreated, and even with proper surgical intervention, a potentially sight-threatening condition may develop in some cases. The most difficult challenges for vitreoretinal surgeons are proliferative diabetic retinopathy (PDR) complicated with tractional RD and proliferative vitreoretinopathy (PVR) developed from rhegmatogenous RD (RRD).PVR is based on the development of fibrocellular membranes on the surface of and under the retina after RRD . PDR is According to our current knowledge, there is no cure or prophylaxis available for PVR yet, apart from the surgical approach . In the Many studies reported an immunological component responsible for PVR and the formation of tractional RD in PDR. In the first studies, only a few proteins could be assayed in one sample by enzyme-linked immunosorbent assay (ELISA) –8. CaepaCurrently, a new technique, the multiplex bead-based immunoassay, provides an opportunity to perform a wide range of molecular analyses in one sample. This helps us understand the interaction among the components of the immunological processes responsible for pathological changes in PDR and PVR , 11. CliThe purpose of this study was to explore the immunological components that are responsible for the proliferative alterations in PVR and PDR and to gain more detailed information and compare the differences in the levels of cytokines, chemokines and growth factors in the vitreous among the different forms of RD.The project was performed under the tenets of the Declaration of Helsinki after approval by the Hungarian Medical Research Council Committee of Science and Research Ethics (No. 15028–2/2017/EKU). All participants gave written informed consent to participate in the study.p = 0.05) was performed and provided the minimum number of eyes to be 65 eyes. The vitreous samples of patients with diabetic retinopathy, RD and ERM were collected for 2 years. As a limited number of samples can be analyzed on one kit of the multiplex chemiluminescent immunoassay, we had to define the maximum number of vitreous fluids. After we analyzed the patients’ data, we excluded the samples that were not matched the standing criteria. We had to exclude a lot of the diabetic samples because of previous intravitreal injections, active proliferation and high glycated hemoglobin.Seventy-three eyes of 73 patients undergoing pars plana vitrectomy were included in the cross-sectional study. Patients were divided into four groups according to the indicated ocular pathology: 30 patients with RRD (without PVR), 16 patients with PVR, 8 patients with PDR and 19 control patients with idiopathic epiretinal membrane (ERM). The exclusion criteria were previous vitreoretinal surgery, penetrating injury, uveitis, aphakia, age-related macular degeneration, and uncontrolled glaucoma. Diabetes mellitus was excluded in the RRD, PVR and ERM groups. In the PDR group, only well-controlled diabetic patients (with glycated hemoglobin [HbA1c] under 8%) were included, and pure tractional RD was present (without vitreous hemorrhage or active proliferation). Previous panretinal photocoagulation was permitted, but intravitreal treatments (steroid or anti-VEGF) were excluded. A priori sample size calculation  was performed by two surgeons in two vitreoretinal centers. Before starting irrigation 0.5 ml undiluted vitreous samples were collected from the eyes. The samples were injected into sterile Eppendorf tubes, immediately cooled to − 20 °C then frozen at − 80 °C within 2 h and stored until the assay was performed.The concentrations of cytokines, chemokines, and growth factors in vitreous samples were measured with a multiplex bead-based immunoassay: Bio-Plex system , molecules were detected with the Human Cytokine Screening Panel, 48-Plex (Bio-Rad Laboratories) after fourfold dilution. According to relevant previous works , 13. TheP-values were calculated via dedicated statistical software . P-values < 0.05 were set to indicate statistical significance.Cytokine concentrations were analyzed with, Kruskal–Wallis analysis of variance and Dunn’s multiple comparison test. Exploratory factor analysis. Since not all correlations are well known among the examined cytokines, exploratory factor analysis was performed with SPSS 23.0 software .An assay could be performed on all samples. Table all RD groups  compared to controls: levels of IL-6 , IL-16 , IFN-gamma , MCP-1 , and MIF  were significantly higher in all RD groups than in the ERM group. The concentrations of IL-8  and eotaxin  were significantly higher in PVR and PDR than in ERM and significantly lower in RRD than in PDR  and PDR . Levels of IP-10 were upregulated in PDR and PVR vs ERM (p < 0.001 both) and upregulated in PDR vs RRD (p < 0.05); however, they were not different in PVR vs RRD. SCGF-beta exhibited the highest expression levels in PVR (p < 0.05 PVR vs RRD) but were not different in PDR vs ERM or RRD. SDF1-alpha was prominent in the PVR  and the PDR  groups.There were four upregulated cytokines in the ups Fig. : CTACK wp < 0.05 PDR vs ERM; p < 0.001 PDR vs RRD and p < 0.01 PDR vs PVR). The vitreous level of IL-18 was found to be elevated in the PDR group compared to ERM (p < 0.01) and RRD (p < 0.05)  Fig. .p < 0.05), IL-17 (p < 0.05) and HGF (p < 0.05) were significantly higher in PDR than in RRD. The concentration of beta-NGF was significantly elevated in PDR compared to RRD (p < 0.05) and PVR (p < 0.05). The levels of MIG were significantly higher in PDR (p < 0.01) and ERM (p < 0.001) than in RRD , IL-7, IL-4, IL-17, RANTES, IL-5, IL-13 and GRO-alpha.Dimension 2: SCGF-beta, SCF, IL-2R alpha and M-CSF.Dimension 3: MIP-1 beta, MCP-3, IL-1ra, MIG and IL-8.Dimension 4: Eotaxin and CTACKDimension 5: IL-18, MIF and IL-16Dimension 6: IFN-alpha2, PDGF-BB and TRAILAs the first step of exploratory factor analysis, variables (molecules) were filtered. Only 29 out of 48 molecules were included in the factor analysis, when valid concentration data were available for at least 70 out of 73 patients. Pearson correlation analysis revealed that a significant correlation was found among all 29 variables. The requirements of exploratory factor analysis were fulfilled: the communality value was higher than 0.3 among all variables, and the Kaiser-Meyer-Olkin (KMO) value was 0.833. The explanatory power of the 6 new variables was 82.36%; therefore, the dimension reductive method could be performed. The 29 variables could be grouped into 6 dimensions:Dimensions mean that the molecules within one dimension exhibit significant activity as individual biomarkers, and their changes are correlated.Factor weights are summarized in Table Our results show that common vitreous biomarkers involved in RDs are IL-6, IL-16, IFN-gamma, MCP-1 and MIF. This finding reveals the strong inflammatory component in the pathology of RD. However, different RD types show a phenotype-dependent profile in the expression of cytokines.The interpretation of our data is challenging due to the complexity of the molecules and RD pathomechanism. Previous studies analyzed similar methods but different aspects of RD. Wladis et al. documented that IL-4, IL-5, IL-6, IL-15, G-CSF, GM-CSF, IP-10, MIP-1 alpha, SCF, and SCGF were significantly increased in PVR compared to primary RRD and ERM . At presAbu El-Asrar et al. evaluated the levels of ten chemokines with ELISA in the vitreous from eyes with RRD, PVR, and PDR, and they came to the conclusion that MCP-1, IP-10, and SDF-1 might be factors in the pathogenesis of PVR and PDR. Our results are consistent with Abu El-Asrar’s, but we could analyze a wider range of molecules in each sample with the help of a multiplex bead-based immunoassay . Wang etTakahashi et al. documented increased levels of IL-6, IL-8, IP-10 and MCP-1 in RRD compared to the control MH group . These rWhen we obtain the result of factor analysis, we focus on which factor and variable coheres with each other. At the same time, we can evaluate the factor weights of the variables to be able to hierarchize the variables of one factor. The bigger the factor weight the more important the variable of one factor, so we can analyze separately how the factor weight develops in a given factor. For example in the group of factor 3 MIP-1beta was the most important element, it had a bigger factor weight than IL-8 Table .To our knowledge, our report is the first to evaluate the concentrations of these 48 cytokines, chemokines, and growth factors in different forms of retinal detachment, including RRD, PVR and PDR with tractional RD. However, our study has some limitations, such as the complexity and high number of cytokines, that need further investigation to detect their relationships more precisely. RD and PDR present with variable clinical features, which might contribute to the multiplex variations in cytokines in fluids. In addition, it cannot be identified whether cytokines are upregulated in the vitreous due to RD (as a consequence) or are already present before detachment (as a causative agent). This limitation is hard to solve due to ethical reasons since the vitreous of healthy human eyes is not accessible in everyday routine clinical care. The exploratory factor analysis highlights some new possible connections and relations among cytokines, which can show some further directions of investigation.We conclude that our results indicate that complex and significant immunological mechanisms are associated with the pathogenesis of different forms of RD, such as RRD, PVR and PDR. Cytokines, chemokines and growth factors are upregulated in the vitreous of eyes with RD, and the upregulation is dependent on the form of RD. The detected proteins are present in different concentrations both in RRD and PVR. In the presence of PVR and PDR, the majority of cytokines are upregulated; thus, they may serve as biomarkers to estimate the progression or severity level of proliferation. Our study adds new biochemical information to previous studies in correlation with proliferative vitreoretinal alterations. More precise knowledge of the levels of vitreal cytokines may represent novel therapeutic targets in the management of these diseases. Future investigations should focus on identifying potential biomarkers to be able to intervene before irreversible proliferative alterations occur.


In college education, it is necessary to participate in social problem solving as well as focusing on academics and creating learning to bring valuable change for the benefit of individuals and society .Service Learning (S-L) is a teaching and learning method to deepen classroom learning through community service or civic engagement . BesidesS-L is an effective educational method, especially for self-efficacy  and citiThe study was conducted on 1,464 female students from Seoul Women’s University, Seoul, Republic of Korea in 2015. The S-L education participation group was 1,184 students (aged 19– 22 yr) and the control group was 298 students (aged 19–22 years).They were informed of the purpose and process of this study and signed the consent form.The S-L course participants needed to spend 32 h performing S-L activities, broken down to two hours per week for sixteen weeks; this also included eight hours of orientation and an introspection conference. The control group was asked not to participate in any S-L program for the same semester. The parameters of civic attitudes and self-efficacy were measured before and after intervention through the 16 weeks.“Civic attitudes” is defined as the responsibility to help others and solve societal problems. The civic attitudes scale was developed by Mabry . Self-efP<0.05.All analyses were performed using SPSS ver. 18.0 . All data are presented as mean ± standard deviation. An analysis of the data was performed using the 2 × 2 (group × time) repeated measures analysis of variance. Statistical significance was set at P<0.001) and self-efficacy (negatively) (P<0.001) (There were interaction effects (time × group) on civic attitudes (positively) (P<0.001) . The stuThus, civic attitudes demonstrated positive improvement. However, the S-L participation group also demonstrated statistically decreased self-efficacy. Self-efficacy is normally increased by the performance of tasks regularly and through the successful completion of homework. Korean university students have, however, normally focused more on studying for college entrance examinations, this has led to a lack of experience in problem-solving in day-to-day life. Since S-L is their first social experience, they focus on the fact that they could not solve a problem rather than that they can solve a problem. Thus, one semester of S-L cannot affect successful experiences enough.Nevertheless, S-L can be a meaningful learning opportunity for students to experience society and introspect. The strategy to encourage students to challenge their volunteer work and learning until they have enough successful experiences is necessary. In this regard, well-designed studies are imperative in the future.


Human creative thinking is unique and capable of generating novel and valuable ideas. Recent research has clarified the contribution of different brain networks  to creative thinking. However, the effects of brain stimulation on brain networks during creative thinking and on creative performance have not been clarified. The present study was designed to examine the changes in functional connectivity (FC) and effective connectivity (EC) of the large-scale brain network, and the ensuing changes in creative performance, induced by transcranial direct current stimulation (tDCS). Fourteen healthy male students underwent two tDCS sessions, one with actual stimulation and one with sham stimulation, on two separate days. Participants underwent tDCS  for 20 min. Before and after the tDCS session, electroencephalography signals were acquired from 32 electrodes over the whole head during the creative thinking task. On FC analysis, the delta band FC between the posterior cingulate cortex and IPL significantly increased only after real stimulation. We also found that the change of flexibility score was significantly correlated with the change in: (i) delta band FC between mPFC and left lateral temporal cortex (LTC) and (ii) alpha band FC between IPL and right LTC. On EC analysis, decreased flow within the DN (from left LTC to right IPL) was observed. Our results reveal that tDCS could affect brain networks, particularly the DN, during creative thinking and modulate key FC in the generation of flexible creative ideas. Science, technology, education, and culture, the things that permeate and enrich every part of our lives, are the products of creativity, which is humankind’s ultimate resource . HoweverCORE) consists of the medial prefrontal cortex (mPFC), posterior cingulate cortex (PCC), and bilateral inferior parietal lobule (IPL) and has been associated with spontaneous generation of creative ideas, as it was found to be involved in internally oriented tasks such as mind wandering, episodic memory retrieval, autobiographical future thinking (MTL), centered around the medial temporal lobe (MTL) and including the hippocampal formation (HF), has also been reported to get activated together with the DNCORE during divergent thinking  also involves semantic processing , the executive control network (ECN), and the salience network (SN) . The relthinking . Takeuchthinking . DNMTL iulations . A numbeulations . When geulations . The midocessing  and playocessing . Previouocessing  and the ocessing . On the ocessing . Additioocessing , maintaiocessing , flexiblocessing , combiniocessing , and novocessing . The salocessing . The ACCocessing . Severalwith ECN  and invowith ECN . Wu et awith ECN . Increaswith ECN .As outlined above, most previous neuroimaging studies focused on correlational methods using electroencephalography (EEG) or fMRI and clarified the contribution of large-scale brain networks to creative thinking. Besides research on the neural correlates of creative thinking, several previous studies investigated the possibility of modulating brain networks related to creativity or enhancing it, using brain stimulation like transcranial direct current stimulation (tDCS)  no history of neurological or psychiatric disease, (2) no history of intracranial metal implantation, and (3) normal or corrected-to-normal vision participated in the experiment. The two participants with the highest sleepiness scores according to the Stanford Sleepiness Scale, assessed during the experiment, were excluded from the analysis considering the possibility that high sleepiness may affect brain activity and task performance during divergent thinking, and make the interpretation of results difficult. Previous studies  have sho∗Power version 3.1.9.7  and conducted two-tailed Wilcoxon signed-rank test. The determined effect size is the median effect size for nominally statistically significant results reported in recent meta-research  applied for 20 min with a 30-s fade-in and fade-out. Previous studies have suggested that cortical excitability is stable for at least 1 h after a 20-min stimulation  connected to a DC-Stimulator 1 × 1 tES device . Electrodes were located according to the extended 10−20 international system for EEG, based on previous reports and the research on cortical projection of EEG sensors . The anomulation ,c. Partimulation . In the The paper-and-pencil version of the alternative uses task (AUT)  was usedThe creativity score of each condition was calculated as follows:(a) Fluency: average number of relevant answers for each everyday object.(b) Flexibility: The judges were instructed to evaluate the number of categories into which the answers for each everyday object could be classified. The average score of five objects evaluated by three judges was calculated as the task score. Intraclass coefficient (ICC) values for flexibility scores of all objects ranged between 0.53 and 0.97 for inter-rater reliability . ICC val(c) Originality: The judges rated the answers for each object regarding their novelty and uniqueness. Specifically, the judges were instructed to evaluate the originality of the answers on a five-point rating scale ranging from 1  to 5   with theAll participants underwent both Real-Stim and Sham-Stim on two separate days, each for 20 min, with an interval ≥3 days between experimental sessions. The experiment consisted of a stimulation session and two EEG recording sessions before and after, with a 5–10-min break between sessions . The EEGElectroencephalography was recorded using the BrainAmp DC (Brain Products) and Brain Vision Recorder (Brain Products) from 32 places over the whole head according to the International 10−20 system  using the ActiCap (Brain Products) with silver-silver chloride active electrodes. During EEG measurements, the electrode located at Fpz was used as ground and the one located at FCz as system reference. The sampling frequency was 500 Hz, the time constant 10 s, and the high cut filter 1000 Hz.Data were analyzed offline using MATLAB 2016b and EEGLAB  version Exact low resolution brain electromagnetic tomography  was analyzed using Lagged Phase Synchronization (LPS). LPS has been widely used to investigate electrophysiological connectivity . Since dxxω, Sxyω, and Syyω represent complex valued covariance matrices, and fxy is the complex-valued coherence. LPS is considered to accurately represent FC, as it excludes the instantaneous phase synchronization due to non-physiological artifacts and volume conduction. The FC between all pairs of ROIs was computed for five frequency bands: δ (0.5–3.5 Hz); θ (4–7.5 Hz); α (8–12.5 Hz); β (13–30 Hz); and γ (30.5–60 Hz).where SEffective directional connectivity (EC) was assessed with isolated effective coherence (iCoh). This EC analysis is performed at the source level, so it requires EEG source localization . Since dKi←j(ω) is the iCoh value at a given frequency ω between ROI i and j, the arrow indicating that j influences i. Ǎ(ω) is the discrete Fourier transform matrix derived by least square fitting of the MVAR model of order p (estimated by the Akaike information criterion). Sε is the covariance matrix of the residual errors of the MVAR model. The LORETA software is able to automatically compute all parameters in this equation, producing an iCoh spectrum as output, when provided with EEG data as input .where as input . In our t-tests were conducted with an alpha level of 0.05. Differences in FC and EC between conditions (Post-Stim vs. Pre-Stim) were assessed in each frequency band with paired t-tests. For the paired stimulation type comparison, (A1–A2) = (B1–B2) design was used, where A1 and A2 stand for the Real-Stim  and B1 and B2 stand for the Sham-Stim . Randomized statistical nonparametric mapping (SnPM)  was determined as corresponding to a p-value of 0.05. We also performed Spearman correlation analysis between the LPS changes of 275 FCs (5 frequency bands ∗ (11∗10/2) connectivities) and the change in score of the three dimensions of creativity. For adjusting multiple comparisons, Benjamini–Hochberg method  for significant testing as well as a false positive rate, q-values in the range of 0.10−0.20 are reasonable in many experiments  on the score change (post-minus pre) of the three dimensions of creativity , two- sided paired g (SnPM)  was applg method  was appleriments  and empleriments .t(13) = −0.23, dz = 6.10e−2, p = 0.82 ; flexibility: t(13) = −0.33, dz = 8.85e−2, p = 0.75 ; originality: t(13) = 0.34, dz = 9.03e−2, p = 0.74 .There was no significant difference between Real-Stim and Sham-Stim types for changes in all creativity scores: fluency: t(13) = 4.50, dz = 1.20, raw p = 5.92e−4, corrected p < 0.05, MPost–Stim = 3.73e−2, SDPost–Stim = 5.15e−4; MPre–Stim = 1.59e−2, SDPre–Stim = 9.88e−5] was observed  and r-IPL  . However, in the paired stimulation type comparison, significantly decreased low γ-band  flow from l-LTC to r-IPL was observed  . No signCORE) and PCC (DNCORE) and decreased the low γ-band EC from l-LTC (DNSUB3) to r-IPL (DNCORE) during divergent thinking. Creativity performance  was not significantly affected by the stimulation, but (i) the change of δ-band FC between mPFC and l-LTC; and (ii) the change of α-band FC between r-IPL (DNCORE) and r-LTC (DNSUB3) induced by tDCS are reflected in a change in creative thinking flexibility. The present study is the first to investigate the effects of tDCS on brain networks (EEG-index) related to creative thinking, and their causal relationships with creative performance.Here, we investigated the effects of tDCS (anode over l-DLPFC and cathode over r-IPL) on brain networks during creative thinking and the causal relationships between the changes in connectivity of the large-scale brain network related to creative thinking, and changes in creative performance. We found that applying tDCS increased δ-band FC between r-IPL  to the r-IPL (DNCORE) significantly decreased in Real-Stim compared to Sham-Stim. This effective connectivity has not been reported in the previous creativity research, but considering that the change in FC between (i) mPFC(DNCORE) and l-LTC(DNSUB3); (ii) r-IPL (DNCORE); and r-LTC (DNSUB3) showed a strong positive correlation with the change in flexibility score, this decreased flow may have causally affected these FCs within the DN and then affected the flexibility of the output.The r-IPL and PCC both belong to the DNognition . The r-Iognition . A previognition . TherefoDUB3 and the exact anatomical region selected as ROI in this study is the middle temporal gyrus (MTG). The left-MTG plays a key role in semantic processing  and r-LTC (DNSUB3) also positively affected the flexibility of divergent thinking. Several previous results support the important role of the right MTG for insight (SUB3) in distant semantic activation and the role of r-IPL (DNCORE) in internal oriented cognition, the strength of the functional connection between these two DN regions may causally affect the flexibility of creative thinking.In addition, the change of α-band FC between r-IPL (DN insight . When pe insight . “Semant insight . During This study has some limitations. First, the sample was small and biased, for instance, in terms of sex and age range, which may affect the robustness of results. Second, there is no control stimulation in terms of polarity or region. Therefore, it is difficult to definitively link our findings to the specific target regions stimulated. Third, the size of the electrodes used for tDCS was relatively large, making it difficult to perform exact and focal stimulation of the l-DLPFC and r-IPL. Thus, the current results should be replicated using more targeted neurostimulation techniques, such as High-Definition tDCS . Fourth,The current study used connectivity analyses to investigate the effects of tDCS on brain networks during divergent thinking and the associated change in creativity performance. Our findings provide new evidence regarding the neural mechanism of creative thinking, particularly flexibility. Future research is needed to clarify the causal relationship between mechanism and creative performance, with the aim of devising methods for the enhancement of creativity.The datasets generated for this study will not be made publicly available. Since, we do not have consent of the participants to publish the data, we cannot share the data.The studies involving human participants were reviewed and approved by the Research Ethics Committee of the Graduate School of Engineering of the University of Tokyo. The participants provided their written informed consent to participate in this study.KK, KU, and ZL contributed conception and design of the study. KU and MN supervised the study. KK and ZL conducted the experiment and analyzed the data. KK wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therefore, the goal of the present study was to test two additional factors for PAHCO and subsequently model the second-order structure with two samples from the fields of rehabilitation and prevention. We conducted two questionnaire surveys with persons with multiple sclerosis (n = 475) and teaching students undergoing a basic qualification course in physical education (n = 502). After performing exploratory items analysis, we used second-order confirmatory factor analysis (CFA) and multidimensional scaling to investigate whether the scales could be bundled in accordance with the PAHCO model. The CFAs with 10 factors (42 items) demonstrated a good model fit. In contrast, the second-order analysis with a simple loading structure on the three sub-competencies revealed an unacceptable model fit. Instead, a second-order model variant was preferred  in which body awareness and self-efficacy had theory-conform cross-loadings. The results of multidimensional scaling  were in line with the extracted second-order structure. The present results suggested that the extension of the measurement instrument to 10 first-order factors was psychometrically justified for the two populations. The results from the second-order analyses provided the basis for the creation of sum scores, representing manifest indicators of movement competence, control competence, and self-regulation competence. Future studies are needed that cross-validate the extended measurement model with other populations and that relate the sub-competencies of PAHCO to indicators of health-enhancing physical activity.The physical activity-related health competence (PAHCO) model assumes that individuals require movement competence, control competence, and self-regulation competence to lead a healthy, physically active lifestyle. Although previous research has already established some measurement factors ( As a rather “qualitative”  or not confirming the existence of an official medical diagnosis of MS (n = 3). The remaining 475 participants were predominantly female (73.5%), on average 47.8 ± 10.0 years old, and had a body mass index (BMI) of 25.2 ± 5.6 kg/m2. The included pwMS had a mean patient-determined disease steps (PDDS) value of 2.74 ± 1.96, with their first official diagnosis being made 15.22 ± 9.27 years ago. Among the participants, 61.9% were undergoing immunotherapy and 62.7% were still employed.The previous step within the successive validation and assessment development strategy  was condHauptfach), as a secondary, minor subject (German: Didaktikfach), or as a third, subsidiary subject . The basic qualification program is the compulsory course for all elementary, middle, and special education teacher candidates who chose physical education as their third, subsidiary subject . Given this preference, it can be assumed that this sample tends to target those individuals who are less interested in or familiar with the topics of PA and health. Within the scope of the PArC-AVE Study , who coordinate the basic qualification programs in physical education at their universities (n = 8), to support the statewide survey in the winter term 2018/2019. All those coordinators were willing to organize the distribution and collection of paper-pencil questionnaires or, if desired, to provide the students with access to an equivalent online survey. This combined assessment strategy led to a final sample of n = 502: ongoing elementary school teachers, 61.8%; middle school teachers, 27.0%; and special education teachers, 11.2%. Two coordinators endorsed the organization via online surveys , while six coordinators preferred paper-pencil variants  to increase the response rate. The participants were predominantly female (87.6%), had a mean age of 23.1 ± 3.7 years, and showed an average BMI of 22.6 ± 3.7 kg/m2.To cross-validate the potential 10-factor measurement model with a different population from the prevention sector, we additionally recruited a sample of teaching students undergoing a basic qualification program in physical education. In Bavaria, Germany, all elementary, middle, and special education teacher candidates must acquire theoretical and practical knowledge in physical education. Depending on individual's educational focus and preferences, physical education can thereby be taken at three different levels, i.e., physical education as a primary, main subject , we relied on robust maximum likelihood estimators (MLR) with Satorra–Bentler scaled statistics to interpret the fit of the models. In addition to the chi-square (SB–χ2) statistics, which tend to systematically reject models of high complexity and models that are tested with huge sample sizes . We also followed suggestions by Hu and Bentler  or satisfactory/acceptable  model fits  and extended the measurement model to a second-level CFA , we successively compared this simple loading model (2) as well as the information criteria by Akaike (AIC) and Bayes (BIC). Furthermore, we transformed the standardized covariance (correlation) matrix with the first-order factors into a distance matrix (with the formula 1–r). Afterward, metric multidimensional scaling (MDS) was performed to more deeply analyze the suitability of a second-order solution and to visually examine the conceptual proximity between the first-order factors. Due to the number of first-order factors (n = 10), we limited the analysis to a two-dimensional (k = 2) MDS solution (Q = 2.25). This decision was supported by the Q coefficient . Accordingly, applying more dimensions would have inhibited the interpretability of the solution. All analyses were run with the software R (Version 3.4.3), including the package Lavaan  and of content-related arguments on the other. As the Mardia test revealed significant violations of multivariate normality , SRMR = 0.032] and five-factor  measurement models on PAHCO. The eight-factor variant, which had recently been worked out with apprentices, showed a satisfactory fit , which also demonstrates the validity of the measurement models for this population. Subsequently, we submitted the five new items of the body awareness factor and the four items of the factor cognitive attitude toward PA to exploratory item analysis. Both factors had good internal consistency with Cronbach's α = 0.93. All five items of the body awareness factor ranged in the middle of the scale (item difficulty: 0.58–0.61), thereby displaying no further statistical anomalies. In contrast, there was high agreement to the items of the cognitive attitude toward PA scale (item difficulty: 0.92–0.93), which was associated with high values for skewness and kurtosis , SRMR = 0.042]. All items loaded significantly (p < 0.001) on their corresponding factor (0.710 ≤ λ ≤ 0.992), and the AVE (0.617–0.906) was good overall (r < 0.834), the Fornell–Larcker criterion was not violated in this sample. The factor reliabilities were consistently located in a good area (0.906 ≤ α ≤ 0.975).There was a good fit for the three-factor  and five-factor  measurement models. The eight-factor measurement model displayed a satisfactory model fit as well . The item analysis with the second sample indicated that the fourth item of the MBD factor was too easy for this population, showing an item difficulty of 0.96 and a kurtosis of 19.98 , SRMR = 0.046]. All items loaded highly significantly on their corresponding factors (p < 0.001). Nevertheless, two items showed low regression weights . This finding was tolerated due to the fact that these items had not disclosed any problems in previous studies and that the corresponding indicator reliabilities (0.37 and 0.38) did not fall in an unacceptable area . The violation of the Fornell–Larcker criterion indicates that the present assessment could not sufficiently discriminate between these two PAHCO constructs in this sample.The sample with the teaching students undergoing basic qualification in physical education also revealed good model fits for the three-factor , with two statistical indicators lying outside the cutoff values (SB–χ2/df > 3.0 and SRMR > 0.10) for acceptable model fits , SRMR = 0.065], and the SRMR especially turned into an acceptable area. The two cross-loadings, both significant (p < 0.001) and substantial in magnitude for a second-order model , contributed to this finding. The pattern revealed that the two loadings even had a stronger conceptual proximity to the second-order cross-factors than to the primary second-order factors , occupying a larger loading in comparison. In the case of the movement competence to body awareness loading, the reduction was considerable, falling below a value of 0.20. Even though such a decrease is considered significant in some recommendations , with the pattern being in accordance with the discriminant validity phenomenon described in the PwMS sample. Nevertheless, the direct comparison between both second-order models  statistically favored the latter variant of the two solutions. The model results are similar when second-order CFAs are computed for the pwMS and teaching student samples separately . Albeit slightly lower in magnitude, movement competence was also strongly associated with self-regulation (r = 0.496) and control competence (r = 0.625).Given the model fit of the second-order structure of PAHCO using the basic 10-factor assessment, we were entitled to create sum scores for the 10 first-order factors and the 3 second-order factors in these two specific populations. The two first-order factors body awareness and self-efficacy, which displayed theory-conform cross-loadings , were inintegrative means that competencies do not refer to comparably isolated and context-independent . Lastly, the PDDS Questionnaire, which provided valuable information for the description of the pwMS sample, has been specifically translated for this study by two independent experts. Future studies should strive for a thorough validation of this instrument.The present study built on previous measurement models on PAHCO  and Friedrich-Alexander Universität Erlangen-Nürnberg, Medizinische Fakultät (467_18B). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.JC defined the validation strategy, developed the new items, initiated and organized the surveys in both studies, performed the analyses, and drafted the manuscript. GS contributed to the item development and refined the manuscript. KP had the major responsibility for both studies and also refined the manuscript. All authors have approved the final version of the manuscript and agree with the order of presentation of the authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Particle size, entrapment, and stability were selected as dependent variables. The obtained formulation containing 25% lecithin, 20% Tween 80, and TPGS 15% fulfilled the prerequisites of the optimum formulation. RO-NTS loaded in situ gel was prepared and optimized for concentrations of Poloxamer 407, and Carbopol, using statistical design. Drug release from in situ gel showed a sustained release profile. The RO IC50 was decreased by half for the in situ gel in comparison to plain RO and RO-EA-NTS. A significant amount of caspase-3 was detected in all the formulation treatments. The studies indicated that EA’s synergistic anti-oxidant effect owing to a high affinity to the PGP efflux transporter and higher penetration in the RO-NTS formulation led to a higher inhibition against human chondrosarcome-3 cancer cell lines. RO-EA NTS–loaded in situ gel had a sustained release that could be significant in localized therapy as an alternative to surgery in the treatment of aggressive tongue carcinoma.The objective of this study was to develop an optimized sustained-release nanotransfersomes (NTS) based in situ gel formulation of rosuvastatin (RO) combined with ellagic acid (EA) antioxidant, to enhance cytotoxic and anti-proliferative activity against tongue carcinoma. The concentrations of lecithin, Tween 80, and  Tongue cancer is a well-known type of mouth cancer. Like other cancer types, it develops as a result of uncontrolled cell divisions, which, in this case, are the squamous cells present on the tongue surface . TroubleStatins are one of the most commonly prescribed drugs all over the world. They are well known for their cholesterol-lowering effect; hence, they are mainly used to treat and protect against cardiovascular diseases . RecentlTransferosomes, defined as vesicular colloidal dispersions composed mainly of phospholipids and nonionic surfactants acting as edge activators (EA), have attracted a great deal of attention in the field of drug delivery . The preEllagic acid (EA) is a polyphenolic phytocompound abundant in natural sources such as green tea, pomegranate, and blackberries . EA has b-poly(propylene oxide)-b-poly(ethylene oxide), are the most commonly used thermosetting polymers and could be applicable for the development of effective thermosensitive drug delivery -2,5-diphenyltetrazolium bromide (MTT) method. Human chondrosarcome-3 cells of density of 5 × 100 values .The caspase-3 enzyme assay was performed for plain RO, RO-EA NTS, and RO-EA NTS–loaded in situ gels. Initially, the cells were cultured in ROMI 1640  and cell extraction buffer was used for further lysis. The collected lysate was diluted with standard buffer as per the assay range for human active caspase-3 traces, and the cells were plated in DMEM (100 μL) at (1.3–1.9) × 10,000 cells/well. Each sample was inoculated in a 96-well plate, 24 h before the caspase- assay . The assp < 0.05.Data were analyzed for multiple comparisons using a one-way analysis of variance  and the level of significance was set at 1); Tween 80 (%) (X2); and TPGS (%) (X3), which were varied at three different levels . Particle size (PS-Y1), entrapment efficacy (EE-Y2), and the stability index (Y3) were studied as responses. The observed values of the responses are shown in From the Box–Behnken factorial design, a total of 17 runs were projected by the design expert for three factors: egg lecithin (%) . The optimized RO NTS formulation was selected based on criteria to attain minimum particle size, maximum EE, and maximum SI, using all the variables within the range as shown in RO-NTS loaded in situ gel was prepared using P 407 and Carbopol 934 P at different concentrations, as shown in The interaction of selected responses and contour plots was shown in The desirability plot for the responses showed a maximum D value of 0.852 that was obtained at optimum concentrations of independent variables. Based on this desirability approach, a formulation containing Poloxamer 407-7% and Carbopol 934P 0.2% could fulfill the prerequisites of optimum formulation .RO EA based NTS were characterized for vesicle shape using a scanning electron microscope. The surface morphology of the prepared formulations was found to be almost spherical with loose aggregates . The vesRO release from the RO-EA, NTS–loaded in situ gel, and the plain RO-EA–loaded in situ gel, was determined using a membrane-less dissolutions model and the results were shown in Stability studies were performed as per Q1A (R2) of the ICH guidelines using RO-EZ NTS and RO-EA NTS in situ gel, which were stored at 4 ± 3 °C and 25 ± 2 °C. Particle size and residual drug content were determined at frequent intervals of 15 days and 30 days as shown in The MTT method was used to evaluate in vitro cytotoxicity against human chondrosarcome-3 cells for optimized formulations of RO-EA NTS, RO-EA NTS–loaded in situ gel, and plain RO after 72 h of treatment. The caspase-3 enzyme assay was performed by inoculating the samples in 96 well plates. A significant amount of caspase-3 was detected in the human chondrosarcome-3 cells in all the treatment formulations, as depicted in 2 value was found to be 0.9689; while the 2FI had R2 values of 0.9219 and 0.8882 suggested for EE and stability index responses. Values of p < 0.05 indicate that model terms are significant. A positive value indicates a synergistic effect, while a negative sign represents an inverse effect [Statistical analysis was performed for the inference of the quantitative effects of the fact factors. The recommended considerable model was quadratic for PS, where the Re effect . 1 (PS) was influenced significantly by (i) the antagonistic effect of A, B , and C (0.0285) and (ii) the synergistic effect of AB with a p-value of 0.0060, being that A effects were the highest. Change in the particle size of transferosomes was primarily due to that a small change or increase in the concentration of TPGS may result in the formation of particles with the desired or minimal size. Variation in the concentration of TPGS will also result in the enhanced entrapment efficiency. In contrast, the only concentration of TPGS  was synergistically affecting the EE%. This result could be due to the Tween 80 or because of the lipophilic nature of the drug, which could enhance the partitioning into the vesicles. The model F-values for the EE and stability index were found to be 63.96 and 43.39, which implied that the model was significant. There was only a 0.01% chance that an F-value this large could occur due to noise. Optimization of the independent variables was done by setting goals for each response then creating an overlay graph [For particle size, based on the ANOVA results , both thay graph . The PS ay graph .2 value was found to be at a maximum (0.9115 and 0.9221), and the quadratic model was suggested for Y2 where the R2 value was found to be 0.9736. Based on the ANOVA results, response Y1 was affected significantly . The model F-value of 62.80 implied that the model was significant. There was only a 0.01% chance that an F-value this large could occur due to noise. Both the selected responses had almost the same magnitude of the synergistic effect on Y1. There would be an increase in the viscosity before gelation with respect to an increase in the concentrations of P 407 and Carbopol 934 P. From the regression equation of Y2; it confirmed that the response was significantly influenced by (i) the synergistic effect of A, B with a p-value of <0.0001, 0.0006, being that Poloxamer 407 % effect was the highest and (ii) the antagonistic effect of A2  [The 2FI was recommended for the Y1 and Y3 responses, where the R <0.001) . GelatioEach response was set for the goals, and the desirability plot for both responses showed a maximum D value of 0.852, which was obtained at optimum concentrations of the independent variables ,41. AccoSurface morphology studies justify the vesicular characteristics with lamellae of the vesicles. They appeared as unilamellar vesicles and were distributed uniformly without any aggregation. Fully saturated polymer and a minimal solvent diffusion rate led to the formation of smooth, spherical, and homogenously distributed particles. Optimized NTS formulation was found to have an average diameter of 76.32 ± 0.0312 nm, which confirmed the monodispersity, thereby indicating mono-dispersity of the formulation . A high Stability studies performed on the RO-EA NTS and RO-EA NTS in situ gel, for particle size and residual drug content, before and after storage, were determined using the same methods given earlier, and at frequent intervals of 15 and 30 days . PercentThe cumulative percentage drug release from plain RO, RO-EA NTS, and RO-EA NTS loaded in situ gels was studied as a function of time. At 2 h, an initial burst release was observed from RO-EA NTS, but at the end of 8 h, 65.14% of the drug was released in contrast to plain RO (10.61%) and RO-EA NTS loaded in situ gels (22.1%). At the end of 24 h, about 65.68% drug was released from RO-EA NTS, which was reduced to 34.3% for RO-EA NTS loaded in situ gel. The drug release from plain RO was very low because of solubility issues. Drug release from RO-EA NTS loaded in situ gel was slower to plateau up to 96 h but the release continued even after 168 h. Nonetheless, there was 98.1% of cumulative drug release from RO-EA NTS by the end of 168 h, in contrast to the RO-EA NTS loaded in situ gel, which had a steady-state release of 59.1% and could still control the drug release even beyond 168 h. The effect was mainly due to the gelling action of P 407 and Carbopol, which were responsible for the controlled release of the drug. The drug release of RO from the formulations was concentration-dependent, but the drug release from RO-EA NTS loaded in situ gel occurred because of diffusion of RO through the water channels of the gel matrix . The iniTotal four treatments  at different concentrations (10–50 µg/mL) were studied for cell viability along with one control group . Figure A novel in-situ gel of rosuvastatin ellagic acid-based nanotransferosomes was formulated and optimized. Based on the desirability approach, a formulation containing Poloxamer 407-7% and Carbopol 934P-0.2% could fulfill the prerequisites of optimum formulation for preparing an RO-EA NTS loaded in situ gel. PDI 76.32 ± 0.0312 nm, confirmed the monodispersity of the formulation. There was better stability of the formulation under refrigerated conditions, as shown in the stability studies. EA loaded formulations showed a considerable increase in apoptosis occurrence through caspase-3-mediation and they also enhanced the tumor suppressor protein levels. EA’s synergistic anti-oxidant effect and higher penetration in the RO-NTS formulation resulted in higher inhibition against human chondrosarcome-3 cancer cell lines. Finally, RO-EA NTS loaded in situ gel may provide a novel strategy (using the deformable vesicular system as carriers) to improve bioavailability, enhance caspase-3-mediated apoptosis, and increase the levels of P21 protein (tumor suppressor protein) in treating tongue carcinomas. This work can still extend to study the in vivo behavior in cancer animal models and permeability studies in cancer tissues.


Atypical or malignant transformation (AT/MT) has been described in WHO grade I meningiomas. Our aim was to identify predictive factors of AT/MT at recurrence. A total of N = 15 WHO grade increases were observed in N = 13 patients . Patients with and without progression at recurrence were similar regarding age, gender distribution, skull-base location, bone infiltration, and Simpson grades. Recurrence-free survival was lower in patients with transformation . Among patient age, gender, skull base location, extent of resection or post-operative RT, no predictor of AT/MT was identified, despite a follow-up of 10,524 patient-years. The annual risk of transformation of WHO grade I meningiomas was 0.12% per patient-year follow-up. Despite the important number of patients included and their extended follow-up, we did not identify any risk factor for transformation. A total of 1,352 patients with surgically managed WHO grade I meningioma from a mixed retro-and prospective database with mean follow-up of 9.2 years ± 5.7 years (0.3–20.9 years) were reviewed. Recurring tumors at the site of initial surgery were considered as recurrence. Whilst their etiology is still not fully understood, it is known that genetic inherited dispositions, hormonal effects, ionizing radiation exposure, as well as immunological factors are implicated in their genesis4.Meningiomas are the most frequent intracranial, extra-cerebral tumors and represent up to 40% of primary intracranial tumors in surgical series5, the management of meningiomas is primarily surgical and aims for a complete/maximal resection of the tumor, including its dural tail. A meningioma management according to the highest standards of care eventually offers high rates of disease-control and globally favorable outcomes9.According to the latest recommendations5 advises a close and diligent post-operative clinico-radiological follow-up, as even long-term recurrences may occur10. The 14 monosomy and 1p36 chromosomal deletion11, the location of the meningioma11. a sub-total resection (STR)11 and higher World Health Organization (WHO) grade13 are amongst the patient-, tumor- and surgery-related factors of recurrence identified and described in the literature.The European Association of Neuro-Oncology (EANO) guidelines14. Although up to 3% are malignant (WHO grade III)16 and 5–7% atypical (WHO grade II), the vast majority of meningiomas are benign (WHO grade I) and show an indolent progression6. Therefore, meningiomas can be perceived as a chronic disease in most of the cases17.The grading of the meningiomas is based on the WHO classification, ranging from grade I to III, with respective overall and progression-free survivals (OS & PFS)21. The overall rate of progression from benign to non-benign histology ranges from 0.16% to 2.0%, according to the most recent data available in the literature25. In the case of LGGs, risk factors for malignant transformation  have been described25. Similarly, age and non skull-base location has been suggested as predictors for atypical or malignant transformation (AT/MT) at recurrence in meningiomas28. This statement is of paramount importance, since patients with recurring meningioma with AT/MT may need more aggressive treatment. Therefore, it is primordial that neurosurgeons and neuro-oncologists are able to identify properly those patients at risk for AT/MT.However, similarly to what can occur in low-grade gliomas (LGG), malignant transformation (MT) has previously been described in formally identified WHO grade I meningiomasIn this study, a large cohort of patients with recurring WHO grade I meningiomas is presented, focusing on the progression toward higher WHO grades at recurrence. Our aim is to identify the predictive factors of AT/MT at recurrence, using a large dataset with an exhaustive and very long follow-up, representing a total of 10, 524 patient-years.Baseline demographics, clinical and radiological characteristics of the study cohort are summarized in Table The distributions of WHO grades at second and third surgery are shown in Table In the WHO grade progression group, no patient had post-operative radiation therapy (RT). In the group with WHO grade stability, a total of N = 28 patients had an adjuvant RT after the first surgery. No difference was observed between the groups, as shown in Table Data on RFS and OS are shown in Table No predictive factor for WHO grade progression at recurrence was found amongst the age, gender, skull base location, the extent of resection as well as the post-operative RT, as shown in Table In this study, we in investigated the risk of atypical or malignant transformation (AT/MT) in recurring intracranial WHO grade I meningiomas, using a database of 1,352 patients treated by surgery with a cumulated total of 10,524 patient-years follow-up. The annual risk of AT/MT was 0.12% per patient-year follow-up, representing approximately 1% of surgically managed patients, and 19.5% of the patients treated for a recurrence.29. Despite the large number of patients included and their long and complete follow-up, no risk factor for transformation among gender, skull-base location, Simpson grade after first surgery, radiotherapy after initial surgery, and advanced age could be identified. We therefore conclude that should risk factors of AT/MT exist, their effect is probably minor.Previous studies have identified advanced age and non-skull base location as predictors for AT/MT in recurring meningiomas5. In the case of WHO grade I meningiomas with documented GTR, the recurrence rate at 10 year ranges from 20 to 39%31. Therefore, an annual follow-up is recommended, up to 5 years after the treatment, then every 2 years. Our results corroborate these data. However, one of our patients had a meningioma recurrence with AT/MT increase more than ten years after initial diagnosis. According to the literature, this is no exception; in their long-term follow-up of surgically managed parasagittal meningiomas, Petersson-Segerlind et al. stated that the 25 years recurrence rate was up to 47%. More specifically, the 10- and 25-years recurrence rates for Simpson grade I–II resections of parasagittal meningiomas were 13% and 48%, respectively. The authors found that the 10- and 25-years mortality rates were as high as 33% and 63%, respectively, of which 50% and 48% of the mortality were directly attributable to the tumor at 10 and 25 years, respectively10. Data on incidentally, observed meningiomas can support these findings. Jadid et al. followed a cohort of consecutive patients referred with incidentally discovered, asymptomatic meningiomas for at least 10 years and found that 35.4% of the tumors showed growth (regardless of tumor size), resulting in a 75% 15-year growth-rate by life-table statistics17.According to the latest EANO recommendations, the post-operative follow-up of meningiomas should be performed by a senior neurosurgeon; the interval between follow-up visits can vary considerably, depending on the Simpson grade, the initial size of the lesion, its location, the age of the patient, as well as its general neurological conditionTo our knowledge, this is the first population-based study assessing AT/MT in originally and formally identified WHO grade I meningiomas using a large cohort with a very long and complete follow-up. Our findings are of paramount importance for neurosurgeons, as they should be able to estimate and inform the patient of the rate of atypical or malignant transformation of meningiomas, that may need an earlier second surgery, but without impact on OS.Our dataset offers a comprehensive and very extended source of data on patients with intra-cranial meningiomas with complete follow-up, since only one patient was lost during the study period.The small number of histological transformations (N = 15 WHO increases in N = 13 patients) limits the finding of statistically associated prognostic factors. However, our cohort is one of the largest, to our knowledge. As WHO classification changed several times over the duration of the study, re-classification of the tumors carries risk of mis-grading, also representing a limitation.33. For instance, McGovern et al.15 found that WHO grade I non-skull base meningiomas have a higher MIB-1 index than grade I skull base meningiomas (1.77 ± 1.86% versus 3.44 ± 3.53%), correlating with lower RFS. Matsuno et al.32 assessed the proliferative potentials of meningiomas using the anti-Ki-67 monoclonal antibody and the MIB-1. The authors observed a statistically significant difference in the MIB-1 index related to gender and age as male patients showed a mean index of 5.5%, versus 2.7% in female patients, and higher MIB-1 indexes were observed in younger patients. Meningiomas with a MIB-1 index ≥ 3% were found to have a significantly high tendency for recurrence within the first 10-year follow-up periods. These results were corroborated by Perry et al.33 Unfortunately, were not able to retrieve information about MIB-1 status of the tumor, as these data were not available for the whole cohort, due to the extended time of recruitment. Certainly, the future will open new perspectives in molecular diagnosis in meningioma management, including the development of biomarkers for AT/MT.The main limitation is the missing of data on molecular characteristics (MIB-1), because of the extended period of inclusion and could not be retrieved for the analysis. The importance of genetics and biomarkers such as MIB-1 and Ki-67 is very well established in the management of meningiomas, at first surgery as well as whenever recurrence occurs34 It is a new yet powerful method for analyzing epigenetic changes in tumor DNA. The analysis of methylation is increasingly used in CNS tumors overall, with excellent diagnostic and prognosis values in terms of biological behavior, open new perspectives towards the identification of predictors of grade changes in initially WHO grade I recurring meningiomas, for which patient-, tumor- and surgery-related characteristics are not sufficient to identify predictors for AT/MT. Unfortunately, we were not able to retrieve information about methylation status of the tumor.Lastly, analysis of the methylation profile has recently been developed and reported by Sahm et al.The annual risk of transformation of WHO grade I meningiomas is 0.12% per patient-year follow-up. Despite the important number of patients included in the study and their extended follow-up, we were not able to identify any risk factor for transformation, among gender, skull-base location, advanced age, Simpson grade after first surgery and first postoperative RT.We performed a review of a cohort of intracranial meningiomas consecutively treated by surgery at Oslo University Hospital (OUH). OUH is a tertiary referral center including two neurosurgical units , covering a total of 3 million inhabitants (representing circa 56% of the Norwegian population). A total of 1,469 consecutive patients were identified from a mixed retro-and prospective database . For this study, only patients with formally identified, surgically managed WHO grade I intracranial meningioma were considered, representing a total of 1,352 patients.30. Gross total resection (GTR) was defined as a Simpson grade I–III resection, according to the latest guidelines of the European Association of EANO5.In most cases, surgery aimed at complete tumor resection, considering both patient and tumor characteristics. Based on surgical reports and the post-operative imaging studies, the extent of resection (EOR) was assessed using the Simpson grade scaleThe histopathological diagnosis of meningioma was confirmed by a senior pathologist on a case-by-case basis. Since the WHO criteria changed during the study period, the tumors included between 1990 to 2001 were re-classified as WHO grade I, WHO grade II and WHO grade III by a neuropathologist .The mean follow-up was 9.2 years ± 5.7 years (range 0.3–20.9 years), with a cumulated total of 10,524 patient-years follow-up. One patient was lost of follow-up due to moving abroad. Regarding the post-operative clinico-radiologic follow-up, patients were generally followed yearly or after 1 and 3 years and then every 5 years. However, the follow-up did not follow a rigorous policy since the initial size of the lesion, its location, the Simpson grading of the resection, the age of the patient and the neurological status were considered on a case-by-case basis.Recurring tumors at the site of initial surgery with radio-clinical correlations were considered as recurrence. Retreatment-free survival (RFS or time-to-retreatment) was defined as the time between the first surgery and the second surgical procedure. Radiological recurrences without clinical impact not requiring any adjuvant treatment, as well as tumors occurring remotely to the primary site, were not considered as recurrence and thus not included in the analysis. In recurring tumors, histo-pathological data was recorded up to the third surgery. In cases where patients were operated more than three times, data on following surgeries were not included in the analysis. As a result, from a dataset comprising N = 1,352 meningiomas, a total of N = 87 and N = 20 patients with first and second recurrence were available, respectively.The study was regulated by the Personal Data Act/Personal Health Data Filing System Act and approved by the Data Protection Official, registered Norwegian institutional review board at OUH (2017/5204). Under this regulation, patient’s written informed consent was not required to collect and analyze data. The study was performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.https://www.r-project.org). The statistical significance threshold was set at p = 0.05. A binomial logistic regression following a general linear model approach was used to identify predictive factors for AT/MT at recurrence. A Kaplan–Meier approach was used to assess the risk of transformation over time.The statistical analysis was performed using R v3.5.1 (


E = 0.001; pS = 0.005). There was no significant difference in PD-L1 mRNA expression between T-OLP and N-OLP (p = 0.128), but the fold-change increase between these groups was significant (Relative Quantification (RQ) = 3.1). In contrast to N-OLP, the PD-L1 protein levels were significantly increased in the epithelial layers of T-OLP (p = 0.007), but not in its subepithelial layers (p = 0.25). Importantly, increased PD-L1 levels were significantly associated to malignant transformation within 5 years. Conclusion: Increased levels of PD-1 and PD-L1 are related to malignant transformation in OLP and may represent a promising prognostic indicator to determine the risk of malignant progression of OLP. Increased PD-L1 levels might establish an immunosuppressive microenvironment, which could favor immune escape and thereby contribute to malignant transformation. Hence, checkpoint inhibitors could counteract tumor development in OLP and may serve as efficient therapeutic strategy in patients with high-risk precancerous lesions.Background: The programmed cell death ligand 1/programmed cell death receptor 1 (PD-L1/PD-1) Immune Checkpoint is an important modulator of the immune response. Overexpression of the receptor and its ligands is involved in immunosuppression and the failure of an immune response against tumor cells. PD-1/PD-L1 overexpression in oral squamous cell carcinoma (OSCC) compared to healthy oral mucosa (NOM) has already been demonstrated. However, little is known about its expression in oral precancerous lesions like oral leukoplakia (OLP). The aim of the study was to investigate whether an increased expression of PD-1/PD-L1 already exists in OLP and whether it is associated with malignant transformation. Material and Methods: PD-1 and PD-L1 expression was immunohistologically analyzed separately in the epithelium (E) and the subepithelium (S) of OLP that had undergone malignant transformation within 5 years (T-OLP), in OLP without malignant transformation (N-OLP), in corresponding OSCC and in NOM. Additionally, RT-qPCR analysis for PD-L1 expression was done in the entire tissues. Additionally, the association between overexpression and malignant transformation, dysplasia and inflammation were examined. Results: Compared to N-OLP, there were increased levels of PD-1 protein in the epithelial and subepithelial layers of T-OLP (p In oral cancer, the programmed cell death ligand 1/programmed cell death receptor 1 (PD-L1/PD-1) signaling pathway represents an important immune checkpoint, which limits immune reactions and contributes to the tumor immune-escape . RecentlThe PD-1 receptor and its ligands PD-L1 and PD-L2 are important modulators of the immune system. The expression of PD-L1 on normal tissues is limited. However, numerous tumor cells overexpress PD-L1 as a strategy to evade immune responses. Thus, the expression of PD-L1 on tumor cells may play an important role in suppressing T cell immune activity and may help malignant cells to escape from the immune system . We demoIn the oral carcinogenesis most OSCC arise on the basis of potentially malignant oral mucosa lesions—oral leukoplakia (OLP) . These aThere is strong evidence that immunologic alterations do contribute to the progression of OLP and occur prior to malignant transformation . OLP witThe immune system plays an important role recognizing tumor- and tumor precursor cells. However, due to immunoediting mechanisms like checkpoints cancer cells are able to escape immune surveillance and establish clinically apparent cancer diseases ,25. PD-1/PD-L1 Immune Checkpoint overexpression is involved in immunosuppression and the failure of an immune response against tumor cells by inhibition of T cell effectiveness. Hence, this mechanism contributes to tumor immune escape and expression analysis of these immune modulators may predict the risk of transformation. There is limited information on PD-L1 and PD-1 expression between OLP with malignant transformation (T-OLP) and OLP without malignant transformation (N-OLP). An immunohistochemical analysis showed an increased PD-L1 expression in T-OLP compared to N-OLP . HoweverThe study aims to analyze the expression of PD-L1 and PD-1 in healthy normal oral mucosa (NOM), OSCC, T-OLP and N-OLP using RT-PCR and immunohistochemistry and to correlate the PD-1/PD-L1 expression values to malignant transformation and infiltration of inflammatory cells in OLP. 2 laser vaporization or observation only. The OLP patients were treated according to the severity of dysplasia as recommended in the guidelines for management of OLP [For this retrospective bicentric study, paraffin-embedded tissue samples were examined. They were taken from historical patient collectives of the universities of Erlangen and Halle  between 1997 and 2015. A sampling technique known as consecutive sampling or total enumerative sampling was used to obtain the samples in both centers. Hence, every subject who met the inclusion criteria of one of the four groups was included in the study until the required sample size was reached. Tissue samples were collected with the consent of the patients and approved by the ethics committee . The specimens were histopathologically evaluated by three pathologists and divided into four groups. Group 1 included tissue samples from patients with oral leukoplakia, which developed into squamous cell carcinoma within 5 years, called T-OLP. The associated samples of oral squamous cell carcinoma were assigned to group 3, called OSCC. Group 2 included oral leukoplakia without transformation within 5 years, called N-OLP. All patients suffering from an OLP were followed up for at least 5 years. The interval between the first diagnosis of OLP and malignant transformation, also called disease free survival (DFS), was determined. Group 4 included samples of NOM. Grades of dysplasia of all OLP (A-OLP) were histopathologically classified into dysplasia grades in accordance with the World Health Organization (WHO) classification of tumors of the Head and Neck 2017 . Accordit of OLP . Thus, lCorresponding OSCC were classified according to tumor size (grouped into T1&2 and T3&4), the state of the lymph node joined together as N0 and N+ to indicate the absence (N0) or presence (N+) of metastases, their differentiation (well-differentiated (G1), moderately (G2) and poorly differentiated (G3)) and their clinical stage (gathered in early (stage I & II) and late (stage III & IV) stages). Lastly, all samples were histologically categorized according to the inflammatory infiltration. They were classified as “none”, “mild”, “moderate” and “severe” based on the sections at 10× magnification according to infiltration density by inflammatory cells in the subepithelium of the affected tissue. Samples that did not present any inflammatory infiltration were categorized as having “no inflammation”. Mild inflammation describes a few, scattered inflammatory cells, moderate pronounces individual inflammatory foci or scattered inflammatory cells, while severe describes several, confluent inflammatory foci or many scattered inflammatory cells in the affected epithelium. Representative examples are shown in The demographic, clinical and histopathological characteristics of patients are summarized in Due to the limited amount of material, not all samples that were immunohistologically examined could be included in the PCR analyses. Therefore, the number of samples and the demographic data of the patient collectives studied by the two methods differ from each other. However, for general statistical analyses concerning localization and inflammation and the association of these parameters to malignant transformation, and the disease free survival , Kaplan–Meier graph and log rank test for survival were carried out with the smaller collective examined by the PCR method.A total of 162 formalin-fixed paraffin-embedded (FFPE) tissue samples were analyzed for PD-L1 expression by real time (RT) PCR. The T-OLP group included 32, the N-OLP group 50 samples. The control groups encompassed 38 (OSCC) and 42 samples (NOM) . All fouTM Kit  according to the recommendations given by the manufacturer. The material tested contained at least 85% of the dysplastic or hyperplastic tissue from oral leukoplakia. The concentration and quality of the RNA was determined using the NanoDrop .The total RNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples using the RNeasy FFPESubsequently, the isolated RNA was translated to cDNA using the Transcriptor High-fidelity cDNA Synthesis Kit according to the manufacturer’s instructions .The cDNA was used to analyze the expression level through real-time quantitative PCR (RT-qPCR). Therefore, specific gene assays were selected from the TaqMan online database using the assay search tool on the Thermo Fischer website for the target genes PD-L1 (Hs00204257_m1) and the house keeping gene GAPDH (Hs02758991_g1). The TaqMan Fast Advanced Master Mix from Applied Biosystems  was utilized for amplification according to the protocol provided. The UNG incubation time was 2 min at 50 °C, the polymerase activation required 20 s at 95 °C, and the PCR (50 cycles) calls for 3 s for the denaturing process at 95 °C and 30 s for the annealing/extending step at 60 °C. GAPDH was used as the endogenous control and the tonsil was applied as a positive control. The samples were analyzed in duplicates. The data were collected and evaluated based on the specifications given by the manufacturer on the ABI Prism 7300 . The average CT-values obtained were used for further evaluations.Samples that were included in the expression analysis show a clear amplification curve for the endogenous control, which a CT value <37. Samples showing a higher CT or no expression for GAPDH were removed from the study, as this indicates the poor quality or lack of quantity of the probe. Samples that received an acceptable CT value for the endogenous control but did not give a CT value for the target gene within the performed PCR cycles were declared as “Non-detects”. There the CT value was determined based on the maximum number of cycles fulfilled, which was 50 in this study . The norSections of 4 µm of the paraffin specimens fixed in formalin were made and then histopathological examination was carried out. The staining for the detection of PD-L1 was performed in the Institute of Pathology at the University Erlangen. After pretreatment in the solution Cell Conditioning 1  for 50 min at 100 °C and incubation for 30 min at 36 °C, the samples were stained with the detection system OptiView BenchMarkUltra  using the antibody DAKO 22C3 .The staining for detection of PD-1 was done in the Department of Oral and Maxillofacial Surgery at the University Hospital Erlangen. After pretreatment in a water bath at 100 °C for 20 min with Antigen Retrieval Buffer 4  followed by cooling at room temperature for a further 20 min, the samples were analyzed for PD-1 expression applying the Anti-PD1 antibody  and the DAKO Detection Kit K5001  according to the manufacturer’s recommendations.®, Budapest, Hungary) for each sample three epithelial and subepithelial image fields  were created. Care was taken to cover the stratifications of the epithelium. Subsequently, the pictures were exported into the TIF format by using the 3DHistech Slide Converter. Then the stains were quantitatively evaluated with the help of the program Biomas . For this purpose, 100 cells of the three ROIs were counted in the epithelia and the positive stained cells were determined. Subepithelially, all cells and the positively stained cells of the image field were counted, so that at least 300 cells were considered. The ratio of positive cells to the total number of cells within the three ROIs was determined and the mean value was calculated from the triplets. This percentage values were used in the statistical expression analysis, now called the labeling index (∆LI).For both antigens membranous staining was defined as a positive result. Representative stains for all immune checkpoints examined are shown in In qPCR analysis the PD-L1 relative gene expression between groups, represented as fold change (FC), were calculated using the ∆∆CT-method. A value greater than 2 is regarded as relevantly increased. The FC in immunohistological staining corresponded to the ratio of mean LI of the groups (∆LI1/∆LI2).p-value ≤ 0.05 was considered statistically significant. The data were additionally examined using receiver operating characteristic (ROC) curves and the corresponding Area Under the Curve (AUC). A cut off points (COP) value was calculated. The COP allows one to derive a collective into two subgroups showing expression of PD-1/PD-L1, either above  or below  the COP. After this subclassification, the chi-square test (χ2-test) was used to explore whether the overexpression of PD-1/PD-L1 was associated with diagnosis, malignant transformation, dysplasia in OLP or TNM classification of OSCC and degree of tissue inflammation. A statistical significance was defined at a p-value ≤ 0.05. Lastly, the positive and negative predictive values were calculated. Additionally, in order to examine the relationship between the expression of the checkpoints and DFS of OLP a Kaplan–Meier graph is added and a log-rank test was made.For evaluation of the results, the statistical software package SPSS 23  was used. It is based on the data collected from the PCR  and the IHC (∆LI). Prior to the statistical analysis, the data were tested for their normal distribution by utilizing the Shapiro–Wilk test. In order to visualize the data box–whisker plots were utilized by displaying the median, interquartile range and minimum and maximum values of the gene expression in the different groups. Non-parametric test were used because the data are not normally distributed. Kruskal–Wallis test and Mann–Whitney U test (MWU) was done to determine whether the expressions between the groups differ significantly in the expression levels of the genes. A p > 0.05). The mean age of patients with T-OLP was marginally higher compared to patients with N-OLP. The samples of corresponding OSCC were obtained from the same patients as in T-OLP group. Subsequently, the mean age of the OSCC group was slightly higher than in T-OLP. The 20 control patients of NOM were significantly younger than OLP and OSCC patients (p < 0.001).The demographic, clinical and histopathological data of the bicentric patient collectives analyzed by different methods are summarized in p = 0.005; In the cohorts analyzed by different methods on 24% up to 29% of T-OLP in contrast to 2% of N-OLP were histomorphologically classified as “high-risk” lesions (D2/D3) (PD-L1 = 8.374). This means that in this group the average expression of PD-L1 was the lowest. T-OLP has the lowest ∆CT average value and hence the highest expression (∆CTPD-L1 = 4.864) followed by OSCC (∆CTPD-L1 = 5.759) and N-OLP (∆CTPD-L1 = 6.474). The expression of PD-L1 mRNA was 3-fold.The mean level of the ∆CT values of the different groups varies . NOM hadThe IHC-determined subepithelial expression of PD-1 showed that the highest LI was given in the group of T-OLP (1.011), followed by NOM (0.66). A low identical expression was found for OSCC and N-OLP (0.2). Elevated mean values were also found in the epithelial compartment for T-OLP and NOM, while N-OLP and OSCC were lower and very similar. Sub- and epithelial expression of PD-1 was approximately 5 times higher in the T-OLP group than in the N-OLP group.The highest epithelial expression of PD-L1 was found in the OSCC group (LI = 10.4), followed by T-OLP (6.1). With an LI of 0.6 epithelial expression of N-OLP was 10 times lower than that of T-OLP. The lowest expression was found in NOM (LI = 0.1). In the subepithelial compartment, expression rates varied marginally. The results are summarized in In order to check the association between expression levels and diagnosis the expression rates of PD-1 and PD-1L in NOM were compared to the OLP and OSCC group.PD-L1 = 11.4, p = 0.0001), N-OLP  and OSCC  in comparison to NOM (p = 0.0001) and NOM, and between T-OLP/N-OLP and NOM (p = 0.0001/p = 0.001)  .p = 0.04) and in OSCC a 99.4-fold increase of PD-L1 was detected. No significant overexpression compared to NOM could be proven for N-OLP tissues . The statistical relevance was confirmed by the AUC values (p = 0.03) and malignancy (p = 0.003). Neither subepithelial PD-L nor PD-1 expression  was significantly changed in the T-OLP group compared to NOM  (PD-L1 = 3.04). That indicates a tendency for evaluated PD-L1 expression in malignant transformation  d. As a rormation .p = 0.007 (p = 0.25)  b. The ex = 0.25) . Additio = 0.25) e. ResultpE = 0.001) and subepithelial layers (pS = 0.005) of T-OLP compared to N-OLP    c. The ov = 0.02) d,e. ResuIn order to examine the relationship between the expression of the checkpoints and DFS time of patients suffering from OLP was calculated. The mean disease free survival of the OLP patients was 77.2 weeks (range: 1–317 weeks). The group of T-OLP was divided in positive (overexpression) and negative (under expression) samples applying the calculated COP for each immune checkpoint. DFS time of the patients in regard to the expression of the different immune checkpoints was assessed . Overexpp = 0.35), PD-1 positive T-OLP neither in epithelial (p = 0.66) nor in the subepithelial (p = 0.51) section or expression of PD-L1 (p = 0.65) in the epithelium  and the differentiation (grading) (p = 0.751), the TNM classification or UICC of the OSCC (p > 0.05). However, there was a significant association of epithelial PD-L1 expression and the severity of dysplasia (pE = 0.005). This could not be observed in the subepithelium (pS = 0.762). UICC stadium (grouped) were related to the PD-L1 expression in OSCC (p = 0.028). Low epithelial PD-L1 levels were predominantly expressed in the early stage than in the late stage lesions. Subepithelial expression was not related (p = 0.647).The overall expression of PD-L1 in the tissues examined by RT qPCR was not associated with the degree of dysplasia (grouped in high and low risk) in OLP . Additionally, no correlation of PD-1 expression and TNM classification and grading could be seen (p > 0.05). The values were also examined for a possible relationship between UICC stage (early and late stage) and expression. With pE = 0.616 and pS = 0.0842 there was no significant association.There was no significant correlation between OLP dysplasia (grouped) and PD-1 expression (p = 0.0001). The grade of inflammation was statistically associated to malignancy (p = 0.0001) and clinical manifestation of an OLP . There was no significant association between malignant transformation and grade of inflammation . However, if OLP were grouped as low and high inflamed tissues the malignant transformation was significantly associated with inflammation (p = 0.035). Results are illustrated in Based on the whole patients collective, distribution of the number of infiltrating inflammatory cells within the groups and its association to malignancy, occurrence of OLP and malignant transformation was evaluated. Out of the T-OLP 3/32 (9.4%) exhibited no, 11/32 (34.4%) exhibited mild, 14/11 (43.8%) exhibited moderate and 4/11 (12.5%) exhibited severe inflammation. Ten out of 49 (20.4%) out of the N-OLP were not, 23/49 (46.9%) were mildly, 13/49 (26.5%) were moderately and 3/49 (6.1%) were severely inflamed. Out of the OSCC all samples displayed inflammation (mild: 7/36 (19.4%), moderate: 23/36 (63.9%) and severe 6/36 (16.7%)), whereas NOM tissues presented only mild (16/35 (45.7%)) or no inflammation (19/35 (54.3%)). The χ square test revealed that the grade of infiltration with inflammatory cells was significantly increased in OLP and OSCC compared to NOM (p= 0.0001). The expression rates rose with the increasing number of inflammatory cells (p = 0.047). If OLP were grouped as low and high inflamed tissues expression was also significantly increased . Epithelial expression was statistically correlated in all comparisons of inflammatory infiltrates except between no inflammation and mild inflammation (p = 0.071) (p = 0.33) nor subepithelial (p = 0.07) could be demonstrated (data not shown). The same results were obtained after grouping the samples in high and low inflammation. Only PD-L1 was significantly overexpressed in the epithelial portion of highly inflamed tissues compared to not or mildly inflamed ones  . No statp = 0.04). Statistical analysis using the Kruskal–Wallis test showed a significant relationship between overexpression of PD-1  (data not shown). These results could be confirmed by the MWU test if the cases were divided into lowly and highly inflamed OLP tissues (p = 0.003) of the OLP but not for the subepithelial one (p = 0.576). After grouping into low and highly inflamed tissues, a significant association with the increased expression of PD-L1 in the epithelium except between none and mild inflammation was confirmed  revealed that 50% (24/48) of the T-OLPs were highly inflamed, whereas only 30.2% (16/53) of the N-OLPs did so. The association of malignant transformation and inflammation was significant ( tissues . A signionfirmed .p = 0.003)  .p∆PD-L1 = 0.32, PPD-1_E = 0.37, PPD-1_S = 0.75, PPD-L1_E = 0.55, PPD-L1_S = 0.37). If the different localizations were compared to each other, the MWU test showed no difference in the expression levels of the immunomodulators tested between the groups, neither in the PCR nor in the immunohistochemically investigation (data not shown).The Kruskal–Wallis test revealed that the expression rates of the immunomodulatory actors were not significantly changed in regard to the localization of the specific OLP  display a significantly increased PD-1 expression compared to N-OLP. This was observed in the epithelial and in the subepithelial compartment. Activated T cells show an upregulation of their PD-1 expression . The PD-Previous data show that T-OLP has an increased macrophage density and a shift towards the immune-tolerant M2 polarization of macrophages . These dWith regard to immunosuppressive checkpoint ligands, an increased expression of PD-L1 was detected in the epithelial compartment of T-OLP compared to N-OLP in immunohistochemistry. In the subepithelial compartment, no significant differences in PD-L1 expression rates between both OLP groups were seen. Interestingly, the RT-PCR analysis revealed no significant difference in PD-L1 mRNA expression between T-OLP and N-OLP. This could be explained by the fact that epithelial and subepithelial tissue was included in the RT-PCR analyses. This underlines the need for distinguishing different tissue compartments  when investigating immunologic parameters. This can be facilitated by a differentiating immunohistochemical approach. Additionally, OLP with a high degree of dysplasia also showed an increased degree of PD-L1 expression. This indicates, that an accumulation of genetic aberrations in oral epithelial cells that occurs during malignant transformation might be accompanied by an increase in PD-L1 expression.PD-L1 is expressed by antigen presenting cells like macrophages but also by tumor cells . In the Cancers are caused by somatic mutations that can result in the expression of tumor-associated antigens that drive tumor progression and might also contribute to tumor initiation. These immunogenic neoantigens lead to specific T cell responses . AdditioLenouvel et al. found that increased PD-L1 expression was associated with the epithelium adjacent to tumor invasion. The presence of PD-L1 in the adjacent epithelium suggests that it may have been present before invasion and could have played a role in cancer development and progression. Hence, they suggest that the presence of the immune checkpoint protein in the adjacent non-tumor epithelium could be an indication of its participation in early carcinogenesis and metastasis . PD-L1 eBy determining a cut-off point (COP) for PD-L1 and PD-1 expression and allocating the individual cases to the positive (increased expression) and negative group, the value of both parameters as a predictive marker for malignant transformation of individual OLP cases could be evaluated. The χ2-test revealed that PD-L1 and PD-1 could serve as predictive markers for OLP malignant transformation with acceptable sensitivity and specificity. Additionally, DFS time of patients of the T-OLP group who were positive for epithelial overexpression of PD-1 and PD-L1 was shortened. However, Kaplan–Meier graphs and log-rank-tests revealed no significant association between DFS time and positivity for immune checkpoint expression. This could already been shown for tumors, where the PD-L1 staining was not significantly associated with DFS . Hence, Therefore, PD-L1 and PD-1 should be evaluated as predictive parameters in prospective analyses. However, it needs to be considered that other parameters like the melanoma associated antigen A (MAGE-A) expression revealed higher sensitivity and specificity in retrospective studies ,44. There is currently no evidence that any treatment can counteract the malignant transformation of OLP and prevent the development of oral cavity carcinomas . TherefoInhibitory checkpoint molecules are promising targets for cancer immunotherapy. The clinical application of immune-checkpoint inhibitors has also dramatically improved the treatment of patients suffering from OSCC. In this regard the most important immune checkpoint proteins identified are CTLA-4 and the members of the PD-1/PD-L1 axis. In the current study, an enhanced expression of PD-L1 was seen T-OLP and OSCC. Moreover, it was shown that this checkpoint was expressed even higher in T-OLP than in OSCC. Therefore, it could be suggested that the treatment applying anti-immune checkpoint antibodies should be equally effective in treating OLP, as it works to counteract the immunosuppression in the OLP microenvironment and would allow the immune system to get rid of the altered dysplastic cells. This would stop the progression and may even lead to regression of the lesion . The antAn increased degree of inflammation has been associated with the severity of oral lesions. The immunosuppression induced by chronic inflammation was thought to play a role in the progression of OLP to OSCC . The facThe results of this study also support the hypothesis that a higher inflammation rate is related to malignant potential, as most of the OSCC and more than half of the T-OLP have moderate to severe inflammation. The association between the malignant transformation and the degree of inflammation was statistically significant. Moreover, a statistically significant association was found between the expression of PD-L1/PD-1 and the degree of inflammation. These findings are in accordance with previous studies that postulate the importance of inflammation as a factor in carcinogenesis . In summIt is well established that the risk of malignant transformation is significantly associated to the localization of OLP. Thus, OLP, which developed on the tongue or at the floor of mouth progress more often into malignancy as OLP at other sides of the oral cavity . AdditioSmoking and alcohol abuse drastically increase the risk of malignant transformation. Hence, these habits have to be taken into account when assessing the risk of malignant transformation. These data were not collected in this study. This could be seen as a shortcoming of the investigation. However, the present study was only intended to show the influence of the PD1/PD-L1 axis as an immunosuppressive agitator on malignant transformation and whether the dignity of a potentially malignant lesion can be better assessed by detecting overexpression of these genes. This question was investigated independently of consumer behavior. Nevertheless, these parameters will be considered in our further multicenter study.Due to the limited material, it was not possible to perform expression analyses for all checkpoints in each sample. For this reason, the cases included in the expression analyses vary greatly. This could reduce the significance of the results. In our planned prospective study, this pitfall will no longer occur, as the material will be used exclusively for the expression studies with the selected markers and all samples taken from patients will not only be fixed and embedded in formalin, but also archived as tissue samples in RNA later.Markers of T and MDSC cells may help to elucidate the status of immune cells. However, we did not perform any expression analysis for CD8 until now. However, we are going to carry out a study, which aims to investigate the expression of several T cell  and MDSC markers  in T-OLP and N-OLP.All over, the number of samples included in the analyses limited our findings particularly for survival. Further studies are needed to confirm the results. However, we are confident we reached a sufficient sample size in our started predictive study to clarify the prognostic impact of checkpoint expression.The data indicated that upregulation of PD-L1 may be associated with disease progress in oral potentially malignant disorders and malignant lesions. A high degree of inflammation in OLP was associated with high expression of PD-L1, PD-1 and malignant transformation. A differential immunohistochemical approach is helpful for the analysis of immune cells in cancer precursor lesions.PD-1/PD-L1 may represent a prognostic indicator to determine the risk of malignant progression of OLP. Local immunosuppression in the epithelium could be induced by PD-L1 overexpression in epithelial cells and trigger malignant transformation. Hence, checkpoint inhibitors could counteract tumor development and serve as therapeutic agents in patients with high-risk OLP lesions.


Although the World Health Organization has recommended the diagnosis and prophylactic treatment of latent tuberculous infection (LTBI) in child household contacts of tuberculosis (TB) cases, the national programs in high-burden TB regions rarely implement adequate screening of this high-risk group, mainly because of resource limitations. We aimed to evaluate the prevalence of LTBI among pediatric household contacts of TB cases in two high-burden provinces in Iran.We conducted a cohort study in children who had been in household contact with a TB index. All subjects were assessed for active TB disease. For LTBI diagnosis, tuberculin skin test (TST) and QuantiFERON®-TB Gold Plus (QFT-Plus) were performed at the time of the index TB case diagnosis, as well as, 3, 12, and 18 months, if the first results were negative. In addition, interferon-γ-induced protein-10(IP-10) concentrations were measured for all participants.A total of 230 children were enrolled, who had contact with an index TB case. Three contacts were diagnosed with active TB. According to the TST/QFT-Plus results, 104 (45.2%) children were identified with LTBI during our study. Significantly increased IP-10 levels were found in LTBI patients compared to healthy contacts. Accordingly, more than 50% of LTBI contacts and about 10% of healthy contacts were considered as IP-10-positive.This study alarmingly illustrates a high prevalence of LTBI among Iranian children exposed to TB cases. We, therefore, emphasize that the children living in close contact with an infectious TB case should be screened effectively and receive prophylactic therapy.The online version contains supplementary material available at 10.1186/s12887-021-02524-3. Mycobacterium tuberculosis (Mtb), is one of the most important causes of childhood morbidity and mortality worldwide : 2134 ± 362.5 for TB1 and 2379 ± 408.5 for TB2) compared to healthy contacts (TST−/QFT-) (Mean ± SEM: 208.6 ± 90.58 for TB1 and 158.4 ± 72.56 for TB2) in response to both TB1 and TB2 stimulation =98.9%), with the highest incidence among children between 5 and 14 years of age . In anotTST is used as the only screening method to identify LTBI children in several high burden TB countries with limited resources, but the low sensitivity of TST, its poor specificity due to cross-reactions with environmental non-tuberculous mycobacteria and the BCG vaccine, as well as its unreliability in children with immunosuppression and malnutrition could limit the applicability of this approach , 32. On P value < 0.0001). Considering the IP-10 results, no significant differences were observed between the TB1 and TB2. Therefore, the IP-10-based assay showed a high discriminating power and could help to distinguish between child contacts with LTBI from healthy contacts. In addition, a high concordance between the IP-10 assay and TST and QFT-plus tests was found.In the present study, we also evaluated the accuracy of IP-10 assay in the plasma of QFT-Plus for the diagnosis of LTBI among Iranian household pediatric contacts of TB cases. The results of some investigations suggest that IP-10 assay could be an alternative to IGRA, may be useful for diagnosing LTBI cases as it would be easier to use , 40. OurIP-10 assay was previously thought to have significant advantages over IGRAs, including the possibility to use smaller blood volumes and to be detected in urine samples. It is also possible to measure IP-10 in dried plasma spots in filter papers allowing cheap and simple mail transportation at room temperature , 41, 42.The advantage of this study is that we used various approach for identification of LTBI among child household contacts, including TST, QFT, and IP-10 assay. Hence, our methodology allowed calculation of the prevalence of LTBI among child household contacts which, so far, has not been performed since LTBI cannot easily be demonstrated using a single method. However, some limitations could be noted in our study. For instance, investigator bias was further limited by the fact that TST could be interpreted differently by independent experts. Moreover, in this survey, some participants enrolled into the study retrospectively and others prospectively, which decreases the power of the study. Finally, in this investigation TST was performed by more than one investigator. Therefore, it might be cause pre-observer bias, which is difficult to objectively evaluate TST results.The results of this study alarmingly illustrate a high prevalence of LTBI among Iranian children exposed to an index TB case. Our experience, therefore, emphasizes that young children living in close contact with a source of TB should receive TB preventive therapy. In addition, establishing a national screening programme and incorporating various methods for LTBI identification among child household contacts should be taken into account in the design of TB control strategies in Iran. Importantly, the identification and prophylactic treatment of LTBI cases is crucial to the control and prevention of pediatric TB and requires more and greater efforts to this end. Moreover, the results of our study, if validated in further large-scale cohort studies, suggested that IP-10 assay could be an alternative biomarker for detection of Mtb infection and might provide some advantages over QFT and TST as the point-of-care test implementation.Additional file 1.